Dispersive Initiation of Replication in the Chinese Hamster Rhodopsin Locus

Dijkwel, Pieter A.; Mesner, Larry D.; Levenson, Victor; D'Anna, Joseph A.; Hamlin, Joyce L.

doi:10.1006/excr.2000.4809

Cited by 36 publications

(36 citation statements)

References 43 publications

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“…In several cases initiation has been shown to occur with relatively low efficiency at many different sites in intergenic regions of the genome (47,48). Recent work suggests that human ORC binds preferentially to AT-rich sequences but otherwise has little sequence specificity (49).…”

Section: Discussionmentioning

confidence: 99%

DNA replication origins in the Schizosaccharomyces pombe genome

Dai

Chuang

Kelly

2004

Proc. Natl. Acad. Sci. U.S.A.

121

135

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Origins of DNA replication in Schizosaccharomyces pombe lack a specific consensus sequence analogous to the Saccharomyces cerevisiae autonomously replicating sequence (ARS) consensus, raising the question of how they are recognized by the replication machinery. Because all well characterized S. pombe origins are located in intergenic regions, we analyzed the sequence properties and biological activity of such regions. The AT content of intergenes is very high (Ϸ70%), and runs of A's or T's occur with a significantly greater frequency than expected. Additionally, the two DNA strands in intergenes display compositional asymmetry that strongly correlates with the direction of transcription of flanking genes. Importantly, the sequence properties of known S. pombe origins of DNA replication are similar to those of intergenes in general. In functional studies, we assayed the in vivo origin activity of 26 intergenes in a 68-kb region of S. pombe chromosome 2. We also assayed the origin activity of sets of randomly chosen intergenes with the same length or AT content. Our data demonstrate that at least half of intergenes have potential origin activity and that the relative ability of an intergene to function as an origin is governed primarily by AT content and length. We propose a stochastic model for initiation of DNA replication in the fission yeast. In this model, the number of AT tracts in a given sequence is the major determinant of its probability of binding SpORC and serving as a replication origin. A similar model may explain some features of origins of DNA replication in metazoans. T he replicon model postulates that initiation of DNA replication takes place at specific chromosomal sequence elements (replicators) that are recognized by regulatory proteins (initiators) (1). This model was originally proposed to explain features of the replication of prokaryotic cells and viruses and has been validated in such systems by a large body of evidence (2). In eukaryotic cells, DNA replication is initiated from hundreds to thousands of different chromosomal sites in each cell cycle, raising the question of whether the replicon model provides a valid description of the initiation process. Although early genetic studies indicated that DNA replication in the budding yeast Saccharomyces cerevisiae conforms to the main features of the model, it is not yet clear that this is the case for most other eukaryotic species.Origins of DNA replication in S. cerevisiae were identified as sequence elements that conferred the property of autonomous replication on extrachromosomal plasmids (3). Genetic analysis demonstrated that such autonomously replicating sequences (ARS) were Ϸ100 bp in length and contained a common 11-bp consensus sequence essential for origin activity, as well as other sequences that augmented origin activity (3-7). The characterization of S. cerevisiae ARS elements led to the identification of the yeast origin recognition complex (ScORC), the initiator protein that recognizes the ARS-consensus sequence (8). The spe...

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Section: Discussionmentioning

confidence: 99%

DNA replication origins in the Schizosaccharomyces pombe genome

Dai

Chuang

Kelly

2004

Proc. Natl. Acad. Sci. U.S.A.

121

135

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“…1A; data not shown). Upon reprobing the transfer for the rhodopsin origin (which resides on another chromosome and, like the DHFR origin, consists of a broad zone of potential initiation sites; Dijkwel et al 2000), the wildtype early-firing pattern of initiation was observed ( 2C). The rhodopsin origin thus serves as an internal control on cell synchrony and the quality of DNA preparations.…”

Section: Dhfr Origin Activity Is Suppressed By a Large ␥-Ray-induced mentioning

confidence: 99%

The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries

Saha¹,

Shan²,

Mesner³

et al. 2004

Genes Dev.

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The dihydrofolate reductase (DHFR) and 2BE2121 genes in the Chinese hamster are convergently transcribed in late G 1 and early S phase, and bracket an early-firing origin of replication that consists of a 55-kb zone of potential initiation sites. To test whether transcription through the DHFR gene is required to activate this origin in early S phase, we examined the two-dimension (2D) gel patterns of replication intermediates from several variants in which parts or all of the DHFR promoter had been deleted. In those variants in which transcription was undetectable, initiation in the intergenic spacer was markedly suppressed (but not eliminated) in early S phase. Furthermore, replication of the locus required virtually the entire S period, as opposed to the usual 3-4 h. However, restoration of transcription with either the wild-type Chinese hamster promoter or a Drosophila-based construct restored origin activity to the wild-type pattern. Surprisingly, 2D gel analysis of promoterless variants revealed that initiation occurs at a low level in early S phase not only in the intergenic region, but also in the body of the DHFR gene. The latter phenomenon has never been observed in the wild-type locus. These studies suggest that transcription through the gene normally increases the efficiency of origin firing in early S phase, but also suppresses initiation in the body of the gene, thus helping to define the boundaries of the downstream origin.

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“…3C was stripped and rehybridized with a probe for the control, early-replicating rhodopsin origin (27), a composite pattern is detected at 90 min consisting of a complete bubble arc and a single fork arc (Fig. 3D) (20). This pattern is characteristic of fragments residing in broad initiation zones that fire in the early S phase (including the wild-type DHFR locus), since such fragments sometimes sustain internal initiation events but more frequently are replicated passively by forks from neighboring initiation sites in the same zone (17,18,20,22,58).…”

Section: Construction Of the Intergenic Ko Cell Lines By The Roko Appmentioning

confidence: 99%

The Dihydrofolate Reductase Origin of Replication Does Not Contain Any Nonredundant Genetic Elements Required for Origin Activity

Mesner

Dijkwel

et al. 2003

Molecular and Cellular Biology

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The Chinese hamster dihydrofolate reductase (DHFR) origin of replication consists of a broad zone of potential initiation sites scattered throughout a 55-kb intergenic spacer, with at least three sites being preferred (ori-␤, ori-␤, and ori-␥). We previously showed that deletion of the most active site or region (ori-␤) has no demonstrable effect on initiation in the remainder of the intergenic spacer nor on the time of replication of the DHFR locus as a whole. In the present study, we have now deleted ori-␤, both ori-␤ and ori-␤, an 11-kb region just downstream from the DHFR gene, or the central ϳ40-kb core of the spacer. The latter two deletions together encompass >95% of the initiation sites that are normally used in this locus. Two-dimensional gel analysis shows that initiation still occurs in the early S phase in the remainder of the intergenic spacer in each of these deletion variants. Even removal of the 40-kb core fails to elicit a significant effect on the time of replication of the DHFR locus in the S period; indeed, in the truncated spacer that remains, the efficiency of initiation actually appears to increase relative to the corresponding region in the wild-type locus. Thus, if replicators control the positions of nascent strand start sites in this complex origin, either (i) there must be a very large number of redundant elements in the spacer, each of which regulates initiation only in its immediate environment, or (ii) they must lie outside the central core in which the vast majority of nascent strand starts occur.In the chromosomes of bacteria, bacterial plasmids, viruses, and lower eukaryotes such as Saccharomyces cerevisiae, replication initiates at genetically defined sites known as replicators, which serve as recognition signals for cognate sequencespecific DNA binding protein complexes termed initiators (34,42). In the chromosomes of metazoans, however, many replication origins consist of broad zones of closely spaced initiation sites, some of which are used with greater efficiency than others (for reviews, see references 14, 19, and 30).An example of the latter is the early-firing Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the 55-kb spacer between the convergently transcribed DHFR and 2BE2121 genes (Fig. 1A) (17,18,22,58). Neutral/neutral (7) and neutral/alkaline (49) two-dimensional (2-D) gel studies on several adjacent and overlapping restriction fragments have demonstrated that there are at least 20 nascent strand start sites (and probably many more) scattered throughout the 55-kb spacer (Fig. 1) (23). However, more-quantitative assays showed that the central 40-kb core, and particularly two subregions within it known as ori-␤ and ori-␥, are preferred ( Fig. 1) (23, 43, 44). The results of an in-gel renaturation analysis of early labeled restriction fragments are shown in Fig. 1A (44), as is the frequency of initiation measured in a quantitative early labeled fragment hybridization (ELFH) assay that used very small origin-containing nascent DNA as a probe (23...

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Dispersive Initiation of Replication in the Chinese Hamster Rhodopsin Locus

Cited by 36 publications

References 43 publications

DNA replication origins in the Schizosaccharomyces pombe genome

DNA replication origins in the Schizosaccharomyces pombe genome

The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries

The Dihydrofolate Reductase Origin of Replication Does Not Contain Any Nonredundant Genetic Elements Required for Origin Activity

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